Heterotopic ossification (HO) is the abnormal formation of bone in structures such as muscle or joints. This devastating complication occurs following major trauma, burns, spinal cord injury/traumatic brain injury, and orthopedic surgery procedures. Patients with HO develop chronic pain, open wounds, restricted joint motion, and nerve entrapment resulting in a significantly diminished quality of life. Patients with HO often require surgical excision, although recurrence and residual deformities are common. Commonly trialed drugs such as bisphosphonates are not tolerated by all patients, have limited efficacy, and do not target the mechanism responsible for HO. Therefore, there is a need to identify solutions to prevent HO in patients. Based on our clinical experience, HO is often preceded by a significant inflammatory insult. We have developed a mouse model of hindlimb tendon transection and 30% total body surface area burn over the dorsum, which consistently produces HO at the tenotomy site. Our evaluation of the tenotomy site revealed substantial macrophage infiltration apparent by 5 days after injury and present in proximity to developing HO even 2 weeks later. Our preliminary findings also show enrichment of a population of osteoprogenitor cells at the injury site present 3 weeks prior to radiographically evident HO. Finally, targeted inhibition of the bone morphogenetic protein (BMP) receptor pathway using small molecule inhibitors significantly decreases HO burden in our mouse model. Through this proposal, we plan to improve prevention strategies for injuries with high HO risk by targeting 1) macrophages or 2) BMP signaling to prevent HO, and the enrichment of osteoprogenitor cells which precedes HO. In Aim 1, we will characterize the contribution of macrophage accumulation to HO using knockout mice incapable of recruiting macrophages to injury sites (Ccr2-null), or by treating mice locally or systemically with inhibitors of COX-1/-2 or TNF?, both of which are involved in macrophage development. We will examine macrophage infiltration using histology, overall HO development using imaging, osteoprogenitor cell presence at the tenotomy site, and osteoprogenitor cell differentiation capacity in vitro. In Aim 2, we will target ALK2; a bone morphogenetic protein (BMP) receptor which we hypothesize is involved in trauma-induced HO. We will use transgenic mice or small molecule inhibitors to target ALK2 locally or globally and examine the effect on HO. We will additionally study the effect on osteoprogenitor cell quantity in vivo and osteogenic differentiation based on in vitro assays. Human tissue in Aim 2 will also be examined for osteoprogenitor cell quantity and in vitro differentiation. Upon completion of this study, we will elucidate how macrophages and/or ALK2 signaling mediate HO formation and whether strategies targeting either of these mechanisms can inhibit HO following trauma.